Liu Xiaoyu, Jin Yao, Li Zhaoyong, Zhao Huashan, Zhou Xinghong, Zhu Yanxin, Gu Yuyan, Zhang Lifang, Zhang Yaxin, He Peikun, Cheng Saibo, Xu Yuling, Jia Yuhua
Pingshan Hospital, Southern Medical University, Shenzhen, Guangdong, 518118, China; Pingshan District Peoples' Hospital of Shenzhen, Shenzhen, Guangdong, 518118, China.
School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong, 510515, China.
J Ethnopharmacol. 2026 Jan 10;354:120525. doi: 10.1016/j.jep.2025.120525. Epub 2025 Sep 2.
Dingxin Recipe III (DXRIII) is a traditional Chinese medicinal formulation that has been employed in clinics for over two decades. It is utilized in the treatment of cardiovascular diseases associated with atherosclerosis (AS) through mechanisms purported to involve the clearing of heat and detoxification, as well as the promotion of blood circulation and the removal of blood stasis. Despite its widespread application and reported therapeutic benefits, its exact mechanisms remain incompletely elucidated.
This study aimed to confirm the anti-AS efficacy of DXRIII and elucidate its underlying mechanism, specifically focusing on the amelioration of endothelial senescence.
The mechanisms underlying the anti-AS effects of DXRIII were elucidated through network pharmacology analysis. In vivo and in vitro models were established using high-fat diet (HFD) induced ApoE mice and HO-induced human umbilical vein endothelial cells (HUVECs), respectively. The histopathologic changes of aortic tissues, inflammatory response, oxidative stress, mitochondrial damage, mitophagy, senescence, and FOXO3a/PINK1/Parkin signaling pathway were evaluated through histological staining, transmission electron microscopy, fluorescent probe staining, RT-qPCR, and Western blot. The constituents of DXRIII were identified via liquid chromatography-mass spectrometry, and the binding affinity and stability of its active compounds with target proteins were investigated using molecular docking and cellular thermal shift assay. Foxo3a was silenced in vitro by lentiviral transfection to determine its involvement in DXRIII-induced mitophagy.
DXRIII effectively ameliorated plaque pathology in AS mice and attenuated endothelial cell senescence. Meanwhile, DXRIII suppressed pro-inflammatory factors and adhesion molecules, mitigated oxidative stress and mitochondrial damage, while activating PINK1/Parkin-mediated mitophagy and upregulating FOXO3a expression. Notably, both mitophagy inhibition Mdivi-1 and silencing of FOXO3a in vitro blocked DXRIII's anti-senescence effects. Furthermore, the active ingredients of DXRIII, including berberine, kaempferol, quercetin and luteolin, showed strong binding affinity with FOXO3a and enhanced its protein stability.
Our findings for the first time demonstrated that DXRIII effectively alleviates endothelial senescence and HFD-induced AS, possibly by activating FOXO3a and subsequently enhancing PINK1/Parkin-mediated mitophagy.
定心方Ⅲ（DXRIII）是一种已在临床上应用二十多年的传统中药制剂。它通过清热解毒、活血化瘀等机制用于治疗与动脉粥样硬化（AS）相关的心血管疾病。尽管其应用广泛且有报道称具有治疗益处，但其确切机制仍未完全阐明。
本研究旨在证实DXRIII的抗AS疗效并阐明其潜在机制，特别关注其对内皮细胞衰老的改善作用。
通过网络药理学分析阐明DXRIII抗AS作用的机制。分别使用高脂饮食（HFD）诱导的ApoE小鼠和过氧化氢（HO）诱导的人脐静脉内皮细胞（HUVECs）建立体内和体外模型。通过组织学染色、透射电子显微镜、荧光探针染色、RT-qPCR和蛋白质印迹法评估主动脉组织的组织病理学变化、炎症反应、氧化应激、线粒体损伤、线粒体自噬、衰老以及FOXO3a/PINK1/Parkin信号通路。通过液相色谱-质谱法鉴定DXRIII的成分，并使用分子对接和细胞热位移分析研究其活性化合物与靶蛋白的结合亲和力和稳定性。通过慢病毒转染在体外沉默Foxo3a，以确定其在DXRIII诱导的线粒体自噬中的作用。
DXRIII有效改善了AS小鼠的斑块病理并减轻了内皮细胞衰老。同时，DXRIII抑制促炎因子和黏附分子，减轻氧化应激和线粒体损伤，同时激活PINK1/Parkin介导的线粒体自噬并上调FOXO3a表达。值得注意的是，体外抑制线粒体自噬的Mdivi-1和沉默FOXO3a均阻断了DXRIII的抗衰老作用。此外，DXRIII的活性成分，包括小檗碱、山柰酚、槲皮素和木犀草素，与FOXO3a表现出很强的结合亲和力并增强了其蛋白质稳定性。
我们的研究结果首次表明，DXRIII可能通过激活FOXO3a并随后增强PINK1/Parkin介导的线粒体自噬，有效减轻内皮细胞衰老和HFD诱导的AS。
